The present invention relates to a method and apparatus for analyzing particles by passing a sample liquid such as urine taken from a subject in a flattened flow, and classifying or counting visible components in the sample liquid by processing the image.
An apparatus is known for taking cells or particles in a flat sheath flow by a video camera, and classifying or counting the particles by image processing. A flat sheath flow passes a particle suspension by covering or surrounding it with a laminar flow liquid. The particle suspension is formed in a flattened flow with a large slenderness ratio.
FIG. 1 is a schematic diagram of such an apparatus, which is intended to analyze components (blood cells, epithelial cells, cylinders, etc.) in a urine sample. The urine sample which is pretreated by dyeing or the like is discharged from a nozzle 12 rate by sample liquid discharge means 18 at a specific flow rate, and is led into a flat sheath flow cell 10. At the same time, in the flow cell 10, a sheath liquid is also fed in by sheath liquid feed means 21, and an extremely flat (thin and broad) sample liquid flow is formed in a rectangular passage 14 with a section having a large slenderness ratio. The sheath liquid is fed by a pump 22 through a sheath liquid chamber 20 (a syringe may be used for feeding the sheath liquid, but the cost is higher). The sample liquid discharge means 18 is a syringe type driven, for example, by a motor 19.
From one side across the rectangular passage 14 (from the back side of the sheet of paper in FIG. 1), a strobe is emitted, and a still image of the sample liquid is taken by a video camera disposed on the other side (not shown). Numeral 16 is a part where an objective lens (not shown) is disposed. The picture which is taken is analyzed by an image processor, and the cell images are drawn, and particles are classified and counted.
When ambient temperature changes, the viscosity of a fluid varies, which affects the liquid flow. In a system where the sheath liquid is supplied at a specific pressure, the flow rate of the sheath liquid varies due to a change in fluid resistance, and the balance of the flow rate of sample liquid and sheath liquid is broken. When the temperature becomes higher, the flow rate increases abruptly.
FIG. 2 to FIG. 5 will be used to explain the flow of sample liquid in the flat sheath flow cell 10. FIG. 2 and FIG. 4 are front views, FIG. 3 is a sectional view of line 3--3 in FIG. 2, and FIG. 5 is a sectional view of line 5--5 in FIG. 4. Numeral 26 shows a sample liquid flow part. Numeral 28 is a video camera taking area.
At low temperature, the viscosity of the liquid (the sheath liquid in this case) is high, the flow rate of the sheath liquid is low, the sample liquid flows in a broad width W1 and a great thickness D1 as shown in FIGS. 2, 3. At high temperature, on the contrary, as shown in FIGS. 4, 5, the sample liquid flows in a narrow width W2 and a small thickness D2.
On the other hand, the taking area 28 is not changed. Therefore, a difference is caused in the volume of the sample liquid that can be taken in the entire sample liquid, which may affect the results of the analysis. For example, the number of taken particles differs.
The ordinate axis of in FIG. 6 denotes the changing rate of the number of taken particles, and the abscissa axis represents the liquid temperature. The peformance is based on the liquid temperature of 24.degree. C. The solid line indicates the change of the number of taken particles in the conventional apparatus, in which the number of taken particles is larger at low temperature, and smaller at high temperature.
In an ordinary flow cytometer, on the other hand, when supplying the sheath liquid at a specific pressure, the thickness of the sample liquid flow varies similarly. As shown in FIG. 7, however, light 30 is emitted by completely crossing the sample liquid flow 32, and the entire sample liquid flow can be detected. Therefore, if the temperature fluctuates, only the frequency band of the particle signals changes somewhat due to flow rate changes of the sample liquid, and serious problems such as change in counting due to change of sample liquid volume to be detected can be avoided.
Thus, in an apparatus design to detect a part, not all, of a sample liquid flow, it is very important to eliminate the fluctuations of flow due to temperature variations.
To solve this problem, for example, in the sheath liquid piping 24 shown in FIG. 1,
(a) a thermostatic unit may be disposed, or PA1 (b) flow rate detecting means may be provided to control the pump pressure.
In the case of (a), a heater or a cooler and its temperature control means are needed, which results in a cost increase and size of the apparatus. Practically, a block of high thermal conductivity in which a flow passage is formed is necessary, and in order to keep this block at a constant temperature, a heater of a larger thermal capacity is required.
In the case of (b), flow rate detecting means and pump output pressure control means are necessary, and the cost is similarly raised. Besides it is difficult to control the pump output pressure precisely.